Random targeting by regenerating peripheral axons compromises sensory and motor function after nerve repair, and limits the usefulness of regenerative prosthetic interfaces after extremity amputation. Regeneration of motor axons to skin provides no function; regeneration of cutaneous axons to muscle degrades sensation. Similarly, poor sensory/motor localization within the proximal nerve stump after amputation defeats attempts to isolate axons corresponding to specific functions. These problems can be overcome by sorting regenerating axons by modality. However, previous efforts to split axon populations have enjoyed limited success for three reasons: 1)There is little comparative data on which growth factors attract only sensory or only motor axons; 2) In the constructs described so far, few regenerating axons are given equal opportunity to respond to both growth factors; 3) We have recently shown that sensory axons adhere to motor axons and limit their outgrowth, a factor not previously appreciated. The goal of this project is to develop an engineering approach that directs axons regenerating from a mixed nerve trunk into discrete sensory and motor channels. These channels could then be used to innervate individual motor and sensory nerves after nerve injury, or to control individual muscles through a regenerative prosthetic interface. The project tests two core hypotheses: 1) It is possible to identify neurotropic growth factors that direct the regeneration of only motor or only sensory axons (Aim I), and 2) Overlapping gradients of these factors can be used to separate regenerating sensory and motor axons (Aim II). These hypotheses will be tested in our organotypic model of mixed nerve regeneration, in which sensory axons expressing tomato red and motor axons expressing YFP are combined within a three-dimensional segment of peripheral nerve. After this nerve is transected, the color-coded axons grow out onto a featureless collagen/laminin sheet, where their pathfinding can be directed by growth factor gradients. These gradients will be established using micro-encapsulated growth factors and bioprinting technology developed in our laboratory. In Aim I we will evaluate the response of sensory or motor axons to candidate growth factors, modifying growth factor concentrations, gradient steepness, and substrate to maximize axon turning. In Aim II we will generate overlapping gradients of the most effective factors from Aim I while blocking the interaction of sensory and motor axons to maximize their turning. The ability to present regenerating axons with overlapping gradients of truly motor-only and sensory-only growth factors in an environment that minimizes axon-axon interactions will thus allow us to optimize separation of these axon populations. If successful, this engineering platform could then be used to construct a three-dimensional prosthesis that separates regenerating sensory and motor axons in vivo, which can be used to improve outcomes in the 50,000 patients/year that undergo nerve repair and the nearly 300 veterans with recent upper extremity amputations.